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To help reverse the recent resurgence of tuberculosis in the United States, use of the most sensitive and rapid diagnostic laboratory tests available that provide reliable results is recommended (1, 8). Currently, these include a liquid and a solid medium for mycobacterial isolation and nucleic acid probes, a chromatographic method, or the BACTEC TB NAP (p-nitro-acetyl-amino--hydroxypropiophenone) test (Becton Dickinson, Sparks, Md.) for identification of Mycobacterium tuberculosis complex (MTBC). In addition, two commercially available nucleic acid amplification tests for direct detection of MTBC are the enhanced Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe, Inc., San Diego, Calif.) and the AMPLICOR Mycobacterium tuberculosis Test (Roche Diagnostic Systems, Inc., Somerville, N.J.), but their use has been limited by the Food and Drug Administration to testing respiratory specimens that are smear positive for acid-fast bacilli (AFB) and which have been collected from untreated patients. The target turnaround time suggested by the Centers for Disease Control and Prevention for detection and identification of MTBC is 21 days or less from receipt of a specimen in the laboratory (8).

Currently the 21-day target for detection and identification of MTBC can only be achieved by use of a nucleic acid amplification test directly from AFB smear-positive respiratory samples or by performing one of the rapid identification tests on growth-positive broth cultures. The latter method, however, occasionally yields false-negative results; i.e., the test is negative for MTBC, which then is later identified by testing colonies on a solid medium (5). When this occurs, the identification of MTBC may be delayed beyond 21 days. An alternative way in which the 21-day target for identification can be met is by performing nucleic acid amplification on aliquots of broth from cultures in which mycobacterial growth has been detected. The AMPLICOR assay has been shown to be a reliable method for identification of MTBC in BACTEC 12B (Becton Dickinson, Sparks, Md.) and ESP II (Trek Diagnostics [formerly AccuMed International], Westlake, Ohio) broth samples (2, 3, 6, 7), but to our knowledge the performance of the MTD has not been evaluated for this purpose. The intent of this study was to examine the reliability of the enhanced MTD, introduced in 1998, for detection of MTBC in positive BACTEC 12B broth cultures.

Culture and identification. Respiratory specimens submitted for mycobacterial culture were decontaminated with 1% sodium hydroxide (final concentration)-N-acetylcysteine and concentrated by centrifugation at 3,000 × g for 20 min, according to standard procedures (4). Approximately 0.2 ml of the sediment was used to prepare a smear for staining with auramine O. To the remaining sediment, phosphate buffer was added to give a final volume of 2.0 ml. For culture, 0.5 ml of the suspension was inoculated into a BACTEC 12B bottle, and 0.2 ml was inoculated onto each side of a Middlebrook 7H11 biplate (Becton Dickinson).

Enhanced MTD. For each of the first 134 specimens, one aliquot was refrigerated and tested by MTD within 72 h and one aliquot was stored for up to 2 months at 70°C for batch testing. Thereafter, both aliquots were stored at 70°C for batch analysis to optimize labor efficiency for the purpose of this evaluation. MTD was performed and interpreted according to the manufacturer's protocol for processed respiratory specimens. Frozen aliquots were first thawed and vigorously agitated with a vortex mixer. Briefly, 50 µl of Specimen Dilution Buffer was added to each Lysing Tube, after which 450 µl of broth specimen, positive cell control, or negative cell control was added to the correspondingly labeled tube. Tubes were vigorously agitated on a vortex mixer for 3 s and then sonicated for 15 min at ambient temperature in a water bath sonicator (Lab-Line Instruments, Melrose Park, Ill.). To labeled amplification tubes, 50 µl of Mycobacterium Tuberculosis Amplification Reagent and then 200 µl of Mycobacterium Oil Reagent were added. Using an extended-length hydrophobically plugged pipette tip, 25 µl of both the sample and control lysates were transferred to the appropriate tube (the remaining lysate was stored at 70°C for further testing, if necessary). Tubes were incubated at 95°C for 15 min in a dry heat block and then transferred to a 42°C water bath for 5 min. To each tube 25 µl of Mycobacterium Enzyme Reagent was added; tubes were capped, gently shaken, and incubated in a 42°C water bath for 30 min. Tubes were carefully uncapped, and 100 µl of Hybridization Reagent was added. Tubes were covered with new caps, vigorously agitated on a multitube vortex mixer (VWR Scientific Products, West Chester, Pa.) for at least 30 s and incubated in a 60°C water bath for 15 min. After hybridization, 300 µl of Selection Reagent was added. Tubes were covered with new caps, vigorously agitated on a multitube vortex mixer for at least 30 s, incubated in a 60°C water bath for 15 min, allowed to cool at ambient temperature for 5 min, and read (in relative light units [RLU]) in a luminometer (Leader 50; Gen-Probe, Inc.). Interpretation of results was as follows: <30,000 RLU, negative; 500,000 RLU, positive; 30,000 to 500,000 RLU, equivocal. For samples yielding an equivocal result, a second aliquot was tested; if the second result was 30,000 RLU, the sample was considered positive, whereas if it was <30,000 RLU, it was considered negative. Results of the enhanced MTD were considered acceptable when the negative cell control was <20,000 RLU and the positive cell control was 500,000 RLU. If the MTD result was discordant from the result obtained by the usual laboratory protocol, the MTD was repeated with the second aliquot and the patient's medical record was reviewed.

Statistical analysis. The Student's t test was used to compare times to identification of MTBC in 12B broth cultures by MTD (i.e., time from bottle inoculation until the culture reached a GI of 50 and the aliquots of broth were removed for MTD analysis) and by the usual laboratory protocol. For the usual laboratory protocol, the interval was time from inoculation of a 12B bottle until the GI reached 999, if the probe result for the broth sediment was positive, and time from inoculation until colonies on the original solid medium or on the solid medium to which broth from a positive 12B bottle was subcultured were identified, if the probe result from the broth sediment was negative.